Our long-term goals are to develop a complete understanding of the oligosaccharide chain-cleaving enzymes expressed by Flavobacterium meningosepticum. These enzymes which include peptide-N4(N-acetyl-beta- glucosaminyl)asparagine amidase (PNGase F), glycosyl-asparaginase F (GAase F), and Endo-beta-N-acetylglucosaminidases 1 ,2 and 3 (Endo F1, Endo F2, and Endo F3) are important analytical tools for investigating the structure/function of asparagine-linked glycans in normal and pathological states, and for quality assurance at glycosylation sites of recombinant therapeutic glycoproteins. We seek a comprehensive analysis of these important enzymes including a detailed understanding of their substrate specificity, DNA and protein sequence, as well as their protein structure and mechanism of action. The research is divided according to specific aims that are prioritized into separate but related topics including x-ray crystallographic analysis, chemical and enzymic synthesis of active-center substrates/inhibitors, and molecular cloning experiments. These multiple approaches will converge on a 3-dimensional model which explains binding and catalysis of appropriate substrates, and which predicts future directions for protein engineering and design of these important classes of hydrolytic proteins.